Ex Vivo Plasma Application on Human Brain Microvascular Endothelial-like Cells for Blood–Brain Barrier Modeling
Abstract
1. Introduction
2. Results
2.1. Differentiated BMEC-like Cells Lack Essential Endothelial Cell Markers
2.2. Undiluted Li-H and So-H Plasmas Can Be Applied on BMEC-like Cells Without Altering Viability
2.3. Barrier Properties Improve After Application of Undiluted So-H and Li-H Plasmas Without Significantly Altering Cell Density
2.4. Characterization of Astrocyte and Pericyte Differentiation
2.5. Gradual Assembly of the Coculture Model Confirms Positive Impact of Plasma Incubation and Coculture with Pericytes and Astrocytes on the Barrier Integrity of BMEC-like Cells
2.6. Continuous TEER Analysis Reveal Regenerative Effect of Coculture Under Plasma Treatment
3. Discussion
4. Materials and Methods
4.1. Human Plasma Pool
4.2. Cell Culture
4.2.1. Immortalized Human Cerebral Microvascular Endothelial Cell Line D3 (hCMEC/D3)
4.2.2. Brain Microvascular Endothelial Cell-like Cells (BMEC-like Cells)
4.2.3. Astrocytes and Pericytes
4.2.4. Establishment of a Triple-Culture BBB Model
4.3. Immunofluorescence Staining and Fluorescence-Activated Cell Sorting (FACS) Analysis
4.4. Endothelial Tube Formation Assay
4.5. Plasma and Inflammatory Treatment for Viability, LDH Secretion, and Barrier Analysis
4.6. Viability Analysis and Lactate Dehydrogenase Secretion
4.7. Barrier Functionality Analysis
4.7.1. Transendothelial Electrical Resistance
4.7.2. FITC-Dextran Permeability
4.8. Image Analysis
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BBB | Blood–brain barrier |
BMEC | Brain microvascular endothelial cells |
CNS | Central nervous system |
DAPI | 4′,6-diamidino-2-phenylindole |
ECM +/+ | Endothelial culture medium with FGF and retinoic acid |
ECM −/− | Endothelial culture medium without FGF and retinoic acid |
ECGM | Endothelial cell growth medium |
EDTA | Ethylenediaminetetraacetic acid |
FACS | Fluorescence-activated cell sorting |
FBS | Fetal bovine serum |
FGF | Fibroblastic growth factor |
GFAP | Glial fibrillary acidic protein |
GLUT1 | Glucose transporter 1 |
hiPSC | Human induced pluripotent stem cells |
IL-1β | Interleukin-1β |
IL-6 | Interleukin-6 |
LDH | Lactate dehydrogenase |
Li-H | Lithium heparin |
LPS | Lipopolysaccharide |
NG2 | Neural/glial antigen 2 |
PDGFRβ | Platelet-derived growth factor receptor β |
PS | Pathological stimulation |
S100β | S100 calcium binding protein β |
So-H | Sodium heparin |
TC | Tissue culture |
TNF-α | Tumor necrosis factor α |
TEER | Transendothelial electrical resistance |
VECad | Vascular endothelial cadherin |
VEGF | Vascular endothelial growth factor |
vWF | Von Willebrand factor |
ZO-1 | Zonula Occludens-1 |
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Nelz, S.-C.; Lück, E.; Schölzel, A.; Sauer, M.; Heskamp, J.; Doss, S. Ex Vivo Plasma Application on Human Brain Microvascular Endothelial-like Cells for Blood–Brain Barrier Modeling. Int. J. Mol. Sci. 2025, 26, 3334. https://doi.org/10.3390/ijms26073334
Nelz S-C, Lück E, Schölzel A, Sauer M, Heskamp J, Doss S. Ex Vivo Plasma Application on Human Brain Microvascular Endothelial-like Cells for Blood–Brain Barrier Modeling. International Journal of Molecular Sciences. 2025; 26(7):3334. https://doi.org/10.3390/ijms26073334
Chicago/Turabian StyleNelz, Sophie-Charlotte, Elisabeth Lück, Anne Schölzel, Martin Sauer, Jacqueline Heskamp, and Sandra Doss. 2025. "Ex Vivo Plasma Application on Human Brain Microvascular Endothelial-like Cells for Blood–Brain Barrier Modeling" International Journal of Molecular Sciences 26, no. 7: 3334. https://doi.org/10.3390/ijms26073334
APA StyleNelz, S.-C., Lück, E., Schölzel, A., Sauer, M., Heskamp, J., & Doss, S. (2025). Ex Vivo Plasma Application on Human Brain Microvascular Endothelial-like Cells for Blood–Brain Barrier Modeling. International Journal of Molecular Sciences, 26(7), 3334. https://doi.org/10.3390/ijms26073334